I. Field of the Invention
This invention relates to a novel class of N-protected amino acid-N-carboxyanhydrides and thiocarboxyanhydrides, namely N-urethane protected amino acid-N-carboxyanhydrides and N-urethane protected N-thiocarboxyanhydrides, their preparation and their use in peptide, poly peptide and protein synthesis.
II. Prior Art
Classically, polypeptides of a defined sequence have been prepared by extremely laborious techniques wherein the intermediates have been isolated after the addition of each amino acid moiety. This has complicated the synthesis and made the preparation of long chain polypeptides and proteins nearly impossible because of low yields, racemization, and/or other side reaction. In 1963, Merrifield (J. Am. Chem. Soc., 85, 2149) and Letsinger and Kornet (J. Am. Chem. Soc., 85, 2045) suggested the use of insoluble polymeric supports for the growing peptide chain. This process, commonly referred to as a solid phase peptide synthesis, permitted the "purification" of the growing peptide chain, without isolating the intermediate.
Heretofore, the widely accepted methods for both classical (liquid phase) and solid phase polypeptide synthesis required the use of a coupling or activating agent to react with the carboxyl group of an otherwise N-protected amino acid to give a carboxyl-activated N-protected amino acid. This activated species would then be used in several ways to promote peptide bond formation. For example, the activated protected amino acid is allowed to react directly with the free amino group of an amino acid, amino acid ester, or amino acid amide to form the peptide bond. This has been the procedure of choice for preparing peptides for many years. The activation step can be accompanied by a number of possible side reactions. For example, where dicyclohexylcarbodiimide(DCC) is the activating reagent, the active molecule may "re-arrange" to an inactive N-acyl urea.
Another disadvantage of the carbodiimide procedure is the formation of insoluble ureas. This is particularly troublesome in solid phase synthesis and is virtually unacceptable in solid phase flow systems. These ureas also cause difficult purification problems in solution phase reactions.
Researchers have alleviated some of the problems associated with in situ activation by first reacting the DCC activated N-protected amino acid with an alcohol or phenol (such as p-nitrophenol, pentachlorophenol, N-hydroxy-succinimide, etc.) to form an "active ester" which may be isolated and purified and then allowed to couple with the free amine of the next amino acid. This approach is not without its shortcomings, however, because the liberated alcohol or phenol may be involved in or promote other side reactions and active ester couplings tend to be sluggish and require long reaction times.
Another common procedure is to form a "symmetrical anhydride" by allowing two equivalents of N-protected amino acid to react with one equivalent of DCC, filtering the DCU formed and then allowing the "symmetrical anhydride" to couple with the free amine group of the next amino acid. This procedure has the urea problem in addition to requiring use of twice as much of an expensive N-protected amino acid.
Some researchers have recently begun to use carbodiimides that form soluble ureas after coupling, but these are still prone to re-arrangement to N-acyl ureas.
Various types of N-protecting groups have been proposed for use in peptide synthesis, but the most widely accepted class of N-protecting groups are the urethanes. Urethanes are broadly acknowledged to provide a high degree of protection, minimize racemization, are readily prepared, and are stable to storage. Urethane-protecting groups can be prepared which are labile to mild acid (i.e. t-butyloxycarbonyl), strong acid (i.e., benzyloxycarbonyl), extremely mild acid 2(p-biphenylyl)isopropyloxycarbonyl, anhydrous base (i.e., 9-fluorenylmethyloxycarbonyl), and so forth.
Urethane-protected amino acids are commonly prepared by reaction of an alkyl, aryl or aralkylchloroformate (or other suitably activated formate or carbonate) with the amino acid in the presence of alkali metal hydroxide or carbonate in a mixed aqueous/organic solvent system (i.e., Schotten-Baumann conditions). After acidification of the reaction mixture, the urethane-protected amino acid is extracted into an organic solvent leaving all side products in the aqueous phase. After crystallization, these compounds are used for peptide bond formation as described above.
A particularly interesting type of reactive derivative of amino acids for use in peptide bond formation are the so-called N-carboxyanhydrides or N-thiocarboxyanhydrides, such as: ##STR1## wherein R, R' are typically hydrogen or the side chains (or protected side chains) of the common amino acids, and Z is oxygen or sulfur.
Amino acid-N-carboxyanhydrides (it is understood that the term N-carboxyanhydride in this specification and appended claims is to include the N-thiocarboxyanhydrides) are well-known and react readily with most free amines. A primary advantage of N-carboxyanhydrides (NCA's) and even protected NCA's for use in peptide bond formation is the fact that they are potent acylating agents (see Peptides, Vol. 9, page 83). They also generally give higher yields of peptides than DCC or N-hydroxysuccinimide (OSu) ester coupling procedures. But NCA's have not found widespread use in polypeptide synthesis because of the lack of ability to control or limit the coupling reaction. Once an NCA reacts with the free amine of an amino acid, carbon dioxide is immediately liberated and a dipeptide is formed which also contains a free amine. This amine will subsequently react with another NCA to form a tripeptide, and so on. This reaction has allowed amino acid N-carboxyanhydrides to find extensive use in the formation of poly .alpha.-amino acids but has virtually precluded their use in sequential polypeptide formation. Hirschmann, et al (The Controlled Synthesis of Peptides in Aqueous Medium. VIII.) The Preparation and Use of Novel .alpha.-Amino Acid N-Carboxyanhydrides. J.A.C.S., 93:11, 1971, pg. 2746-2774) have succeeded in using amino acid N-carboxyanhydrides for the preparation of di-and tripeptides in aqueous-organic solvent systems by careful control of temperature, pH, salt, and organic solvent of the reaction mixture. However, this procedure is limited to small peptides because of the chemistry of NCA's described above. Furthermore, the products obtained from these solution phase reactions must be extensively purified prior to being used for the preparation of larger peptides.
A variety of N-substituted amino acid N-carboxyanhydrides have been reported in the literature such as N-methyl, N-benzyl, N-acetyl, N-nitrophenylsulfenyl, N-xanthyl, 4,4'dimethylbenzhydryl, trityl, and the like. Several of these substituted-NCA's have been proposed for use in sequential peptide synthesis and particularly for solid phase peptide synthesis, but none have gained acceptance for general use by peptide chemists.
Kricheldorf (Angew. Chem. Acta 85, 86-87, (1978)) proposed the use of o-nitrophenylsulfenyl (NPS) substituted NCA's for use in sequential polypeptide synthesis. These were prepared by the reaction of o-nitrophenylsulfenylchloride with a N-carboxyanhydride in the presence of triethylamine. Subsequently, it has been shown that triethylamine promotes the racemization of NPS-NCA's. In addition, oligomerization due to the action of triethylamine on the NCA, requires that very stringent reaction conditions must be employed (i.e. temperature &lt;0.degree. C. and very slow addition of triethylamine to the reaction mixture) during NPS-NCA synthesis. Halstrom, et al, (Z. Physiol. Chem. 355, 82-84, (1974)) consequently proposed the synthesis of NPS-NCA's by reaction of phosgene with the NPS-amino acid but the yields were very low (about 20%). Once prepared, NPS-NCA's are difficult to store, and tend to "bleed off" the protecting group during condensation, giving rise to multiple coupling, and other side reactions. Also, the nitrogen of the resulting NPS protected peptide possesses substantial nucleophilicity and may undergo additional condensation reactions.
Block and Cox ("Peptides, Proc. of the 5th Europ. Symp., Oxford, September 1962". Pergamon Press 1963, Ed. G. T. Young, pp. 84-87.) proposed the use of N-trityl amino acid-N-carboxyanhydrides for use in peptide synthesis, although they were only able to prepare the simplest N-tritylamino acid NCA's (ie. glycine and alanine). These compounds were prepared by the reaction of a N-trityl-amino acid with phosgene. By this procedure, they were also able to prepare N-acetyl-glycine-NCA. These researchers recognized the potential usefulness of t-butyloxycarbonyl glycine N-carboxyanhydride and benzyloxycarbonyl glycine N-carboxyanhydride but were unsuccessful in their attempts to prepare them and concluded that urethane-protected amino acid N-carboxyanhydrides could not be made? Even if all the N-trityl-NCA's could be prepared it is well known that the use of trityl protection of amino acids in various condensation methods produces low yields because of the considerable steric hindrance imposed by the trityl group. The trityl group is also extremely sensitive to acid which makes the preparation of the trityl-NCA difficult and tends to "bleed off" during normal solid phase manipulations.
Halstroem & Kovacs (Acta Chemica Scandinavica, Ser. B 1986 BYO(6), 462-465 and U.S. Pat. No. 4,267,344) also recognized the advantages and the potential usefulness of N-protected amino acid N-carboxyanhydrides and were able to prepare several N-substituted NCA's which they felt would fulfill all the requirements necessary for use in peptide synthesis. They were able to prepare a number of 9-xanthyl (and related) substituted amino acid N-carboxyanhydrides. These compounds were claimed to be preparable by the direct condensation of xanthydrol with the appropriate NCA in refluxing benzene, toluene, xylene or other alkyl benzene. The water formed during condensation was removed azeotropically. This procedure suffers from the instability of NCA's to heat and to water, consequently leading to low yields and potentially impure products. These compounds may also be prepared by the reaction of phosgene (or phosgene equivalent) with the corresponding 9-xanthyl-amino acid and, in fact, most of the substituted NCA's in this class have been prepared by this procedure.
When used in peptide synthesis the 9-xanthyl-NCA's have been found to react sluggishly requiring as long as 5 hours at 50.degree. C. in solution and 24 hours at 25.degree. C. in solid phase synthesis. This is likely due to the steric hindrance of the 9-xanthyl group and/or the deactivating effect. Another problem associated with 9-xanthyl protection of amine groups is that the nitrogen atom of the 9-xanthyl amino acid formed after the coupling reaction is still nucleophilic and capable of undergoing subsequent condensation reactions. These groups will also tend to bleed off during manipulation. Consequently, to date, substituted amino acid N-carboxyanhydrides of this or any other type have not found widespread use in peptide synthesis, particularly in solid phase peptide synthesis.
Kricheldorf, (Makromol. Chem. Vol. 178, pp 905-939, 1977) has described a method for the preparation of methoxycarbonyl glycine NCA and ethoxycarbonyl glycine NCA. However, Kricheldorf also reports that this procedure was incapable of producing urethane-protected NCA's of amino acids having a side chain other than hydrogen because of steric hindrance.
It is an object of the invention, therefore, to provide the heretofore unobtainable urethane-protected N-carboxyanhydrides and N-thiocarboxyanhydrides of the higher amino acids.
Another object of the invention is to provide procedures for the preparation of pure, crystalline, stable urethane-protected amino acid N-carboxyanhydrides and urethane-protected N-thiocarboxyanhydrides.
Yet another object of the invention is to provide a method for the synthesis of polypeptides utilizing pure, crystalline urethane-protected amino acid-N-carboxyanhydrides, which synthesis offers the following major advantages over conventional methods of polypeptide synthesis:
1) Pre-activation of the carboxyl group to be coupled is unnecessary, thus eliminating side products generated by conventional activating molecules.
2) No additives such as N-hydroxybenzotriazole are needed to inhibit racemization.
3) The only co-product from the coupling reaction is carbon dioxide.
4) These N-protected carboxyl activated amino acids are stable, storable, crystalline materials and, therefore, facilitate and simplify both solid and liquid phase peptide synthesis, especially in automated peptide synthesizers, by eliminating the need for activations, filtrations, and couplings prior to the peptide bond forming reaction. The purification of peptides prepared in solution is greatly facilitated by the use of these novel compounds because of the lack of by-products produced by coupling agents.
5) The procedure will provide, after coupling, the widely accepted urethane-protecting groups on the amino function of the growing peptide chain which may then be manipulated by conventional techniques.